The present invention relates to novel pcptides useful in the amino acid sequencing context and methods of their use. In particular, this invention relates to an internal standard for amino acid sequencing comprising unnatural amino acid residues that is capable of being sequenced simultaneously with an unknown peptide or protein without interfering with the analysis. Further, this invention relates to synthetic control peptides comprising natural amino acid residues that are designed for use in monitoring the proper operation of amino acid sequencers and for confirming that the system properly identifies all the common amino acid residues. These synthetic control peptides can also be used as controls in a wide variety of chemical and enzymatic reactions to monitor cleavage and modification reactions.
Amino acid sequencers typically degrade a protein or peptide selectively and sequentially into amino acid residues, or derivatives of these residues, that arc capable of being qualitatively and quantitatively identified. For instance, the commonly used Edman sequential degradation involves the organic base catalyzed selective coupling of a peptide's N-terminal amino acid with phenylisothiocyanate. The derivatized amino acid is then cleaved from the peptide by treatment with a strong organic acid, typically as an anilinothiazolinone (ATZ) derivative. Repetitive coupling/cleavage cycles at the newly-formed N-terminal amino acid left by the previous cycle provide for sequential separation of the amino acid residues that form the primary structure of the peptide. To determine the identity of the separated derivatives, the ATZ derivative is typically converted to a more stable phenylthiohydantoin (PTH) derivative prior to analysis. These PTH derivatives can then be identified by a variety of analytical procedures, such as by HPLC. The coupling/cleavage cycles, the PTH derivatization procedures and the injection of the PTH derivatives onto an HPLC can be accomplished manually or, more commonly, by fully automated amino acid sequencers as described for instance, in Applied Biosystems User Bulletin Issue No. 14 (Nov. 18, 1985).
Current internal standards available for use in an amino acid sequencer suffer from various disadvantages. For instance, addition of a synthetic PTH amino acid derivative, such as PTH-norleucine, to one of the sequencer solvents is known. This type of internal standard, however, is capable of indicating only that the sample was injected properly onto the HPLC column. A faulty injection step is only one of a multitude of possible malfunctions that could occur during the sequencing process. For instance, the use of a PTH-amino acid internal standard provides no information regarding whether the equipment is capable of actually sequencing a sample. In addition, the PTH-norleucine derivative is unstable and must be added every 2-3 days, making quantification very difficult and wasting expensive sequencer solvents.
Bausch et al., BioPharm 2(5):40-43 (1989) disclose the use of poly-L-ornithine hydrochloride as an internal standard during automated protein microsequencing. The poly-ornithine molecule, which ranges in size from about 15,000 to 30,000 Daltons, degrades to provide a PTH derivative that has a unique chromatographic retention time. Thus, observance of the PTH-ornithinc peak (or lack of the peak) for each sequencer cycle during the sequencing of an unknown protein provides information regarding instrument failure, "bad" reagents and sample-specific problems such as N-terminal blockage. However, because poly-ornithine is a homopolymer, this internal standard is incapable of providing information regarding repetitive yield, which is an important index of sequencer performance. Further, because the PTH-ornithine derivative is produced in each sequencer cycle, this internal standard is extremely susceptible to lag, or carryover from preceding cycles due to incomplete degradation, rendering quantifying the PTH-ornithine peaks relatively meaningless. Additionally, while the use of poly-ornithine as an internal standard provides some information regarding instrument failure for a particular cycle or N-terminal protein blockage, this internal standard is incapable of distinguishing between a blank cycle caused by a missed injection and a blank cycle caused by faulty delivery of chemicals during the sequencer reactions. Without this knowledge, the experiment would have to be repeated, which may not be possible for proteins that are only available in minute quantities. Lastly, the poly-ornithine internal standard is more similar in size to a protein rather than a peptide and, thus, is not as easily washed from the sample support as a peptide sample. Consequently, sequencer conditions which provide for optimized sequencing of the poly-ornithinc standard may not be appropriate for sequencing an unknown peptide.
Thus, there exists a need for an internal standard for amino acid sequencing that does not interfere with the sequencing of an unknown protein or peptide and can distinguish between a blank sequencer cycle caused by the presence of modified amino acids or machine malfunctions, including blank cycles caused by missed injections and blank cycles caused by faulty delivery of chemicals during the sequencer reactions. There also exists a need for an internal standard in which lag does not interfere with subsequent chromatographic peaks and which provides initial yield and several accurate repetitive yields during the actual analysis of the sample unknown. Additionally, there exists a need for an internal standard having a molecular weight similar to peptides to provide a more accurate correlation when sequencing these components.
Mixtures containing stable PTH amino acid residues, N,N'-diphenylthiourea (DPTU), dithiothreitol (DTT) and/or N,N-dimethyl-N'-phenylthiourea (DMPTU) have been used to optimize the separation conditions needed for resolution of the PTH derivatives by the chosen analytical procedure, such as by HPLC. However, these procedures optimize only the final identification step rather than providing guidance for proper conditions throughout the repetitive coupling/cleavage/derivatization/identification cycles. Proteins, such as .beta.-lactoglobulin, have also been used to verify the operation of the amino acid sequencer. However, optimization of the sequencer using high molecular weight components such as proteins can result in inappropriate operating conditions for sequencing lower molecular weight peptides, including conditions which result in the peptide being "washed out" from the glass filter disc of the amino acid sequencer. Thus, even though .beta.-lactoglobulin contains appropriate amino acid residues suitable for at least three determinations of the repetitive yield, these repetitive yield values may be inapplicable for peptide unknowns. Further, no single peptide is available that has even a few of the uncommon or difficult to measure amino acids sufficiently close to the N-terminus to provide for sequencer optimization that takes into account these residues while still providing information regarding the common amino acids. Thus, there exists a need for a synthetic control pcptide, or a mixture of synthetic control pcptides, capable of being used to monitor the proper operation of an amino acid sequencer so as to allow optimization of the sequencer with respect to the sequencing of peptides. In particular, there exists a need for control peptides designed to monitor the sequencing of the common amino acids as well as the rarely seen or difficult to measure amino acids in addition to providing an appropriate residue structure and sequence to allow accurate determination of lag and repetitive yield.
While it is possible to use existing proteins and peptides as controls for chemical and enzymatic reactions, no polypeptide is available that is suitable for a wide variety of cleavages or reactions. Furthermore, because proteins contain many cleavage sites, use of proteins to monitor these reactions results in far too many fragments, which yield complex chromatograms. Thus, monitoring these reactions by use of a control protein unnecessarily complicates the subsequent analysis, making it difficult to determine the products and the reproducibility of the reaction. In addition, the commercial preparations of proteins or peptides often vary in purity and some residues may be modified in variable amounts in different preparations or from different manufacturers. Thus, there also exists a need for synthetic control peptides having amino acid sequences designed to have a limited but sufficient number of the appropriate amino acid residues so as to allow the monitoring of a wide variety of chemical and enzymatic reactions.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.